Glycosaminoglycans (GAGs) are polysaccharides composed of a repeated disaccharide subunit and known to be important for the maintenance of structure and mechanical properties of the extracellular matrix. More recently, changes in the content, size, and composition of GAGs have been found to play biological roles in disease processes as well. For example, chondroitin sulfate (CS) is the most abundant GAG; however, only the special form of CS, chondroitin 4-sulfate, is produced by cancer cells and is required for cellular attachment, migration, and invasion of tumor cells. The most studied GAG, hyaluronan (HA), has been found elevated in many inflammatory diseases. Recent works suggest not only the amount, but the size of the HA polymers is also altered in pathological states. The molecular weight and disaccharide subunits of GAGs are highly heterogenous and as such, there is increasing interest in assigning different biological functions to different molecular weight GAGs. For example, it was found that the low molecular weight component of serum HA can be used to differentiate metastatic from non-metastatic breast cancer. Even with these exciting new discoveries of GAG functions, GAGs are still less studied and understood than their proteoglycan partners due to a lack of convenient research tools. Sulfated GAGs are usually covalently linked to a core protein and form a complex called proteoglycan, and an even larger complex can also be formed by multiple proteoglycans. This multi-dimensional complex makes isolating GAGs from their core protein critical in accurate GAG detection. GAG isolation is a laborious and time-consuming process involving days of protease digestion, chloroform extractions, dialysis, and ethanol precipitations which do not fully remove impurities. In addition, many established GAG detection methods require enzymatic digestion in addition to GAGs isolation making such methods unable to detect intact GAGs (size determination). The primary goal of this project is to develop robust and accessible research tools enabling widespread investigation of GAGs? roles in pathological process. This Phase I project will create simple-to-use tools for analyzing GAGs from complex biological samples; and make these tools available to any lab with common biology equipment resulting in a significant savings of time and labor. Aim 1 will develop an easy-to-use GAG prep kit via selective high-throughput mini-chromatography, aim 2 will focus on development on straight-forward homogenous intact GAGs assays. The developed tools will then be validated in aim 3, using breast cancer cell lines and patients? serum. These tools will enable new explorations in GAG-based cell signaling, drug development, and biomarker discovery.